In this article we will discuss about the role of Phytochemistry in systematic botany.
Phytochemistry (plant chemistry) also plays an important role in systematic botany. This is based on the assumption that related plants have a similar chemistry.
For example, In Lichens chemical methods are used for identification of genera and species. But there is a controversy between Taxonomists as some morphologically similar lichens are chemically different; these two strains are then known as “chemical strains” and not as different species. In recent past at least 80 different lichen substances are identified based on the chemistry of lichens.
Chemical substances found in plants are Betalins, Anthocyanins, Glucosinolates, fatty acids and oils, flavonoides etc. There are three major techniques to study the phytochemical substances.
These are as follows:
(A) Serology,
(B) Electrophoresis, and
(C) Amino acid sequencing.
(A) Serology:
Serology is the branch of biology related with the nature of antigenic materials and antibodies. Serological data help in detecting and comparing non- morphological characteristics in taxonomic studies.
Serology is one of the techniques which is used in chemosystematic studies. It relies on the immunological reactions shown by the mammals when they are invaded by foreign proteins or antigens. The phenomenon was first used in 1897. Carl Mez and his associates at Korigsberg in Germany used serological bioassay methods to study angiosperm family relationship.
The fundamental principle of serology is that when plant extract containing antigen (protein) is injected into a mammal, a proteinaceous antibody will be formed in the animal. This antibody is specific to the antigens (E) and capable of coagulating it.
These antibodies are extracted from the blood in the form of antiserum. Antiserum coagulates further supply of antigen and thus can be used as a standard test against plant extracts. The amount of coagulation is used as a measure of similarity to plant extract. Hence, the similarity of the species corresponding to the plant extracts (E1, E2…) will be compared.
The individual antigen antibody reaction, forming the total precipitation can be recorded separately in two ways:
(a) By following the antigenic material and anti serum to diffuse towards one another in a gel (different proteins travel at different rates so different reaction will take place at different sites in the gel) this method is known as double diffusion serology.
(b) By first separating the antigen unidimensionally in a gel by electrophoresis and then allowing them to diffuse towards a trough of antiserum. This method is called Immuno electrophoresis.
Presently in the Double diffusion serology method the antiserum is placed in a small circular well, surrounded by a ring of similar wells containing the samples of antigens. Radio Immuno assay (RIA) and Enzyme Linked Immunosorbent Assay (ELISA) are also used where either the antibodies or the antigens are labelled with radioactive molecules or enzymes which can be detected in even very small quantities.
Most of the serological work is carried out on organs where protein is found as a food reserve, e.g., seeds, stems, tubers etc.
Serology is a useful taxonomic tool at all levels from above the family to below the species. Hawkes worked on the relationships of the tuber bearing species of Solanum to elucidate the ancestors and close relatives of the cultivated variety of potatoes. Jensen worked at family level on Ranunculaceae and Berberdaceae.
The classification of Ranunculaceae at the tribal and generic levels indicated by serological relationship strongly supported that based on classical evidence particularly from the chromosome. Fairbrother worked on Cornaceae and Magnoliaceae.
Smith has the relationship of annual brome grass (Bromus), relating immuno electrophoretic data to these from morphology, cytology, and cytogenetics. He recognised a variety of Bromus secalinus as a new species. B. pseudosecalinus on the basis of his studies.
Serology is not useful in identification of particular proteins. It cannot interpret the significance of the results.
(B) Electrophoresis:
Electrophoretic separation of protein is dependent on the amphoteric properties of amino acids. Whereby they are charged negatively or positively according to the pH of the medium and will travel through a gel at various speeds in a voltage gradient. The principle is basically applied and carried out in a polyacrylamide gel. Protein separation depends upon the sieving effect of the gel pores.
A crude separation is brought about in the larger porous zone while a complete separation into discrete bands in the smaller pored zone. After suitable separation time, the current is switched off and the separated polypeptide on the gel are located by stains. General protein stains may be used or specific stain is used for a particular enzyme.
The SDS-PAGE System:
Polyacrylamide gel electrophoresis when carried out in the presence of SDS – (Sodium Dodecyl Sulphate) the whole system is called as SDS-PAGE system, i.e., sodium dodecyl sulphate polyacrylamide gel electrophoresis system.
SDS is considered as a strong detergent which can denature proteins. Under appropriate conditions all reduced polypeptides bind the same amount of SDS on a weight basis, i.e., 1.4 gm SDS/gm of polypeptide. The separation is dependent on the size of the SDS—polypeptide complexes.
SDS -PAGE may be run on either a disc or a slab gel system. Most widely used is slab gel system because:
(a) Versatility of manipulation as drying, photography etc.
(b) Uniformity with regard to polymerisation, shrinking, swelling during staining and destaining.
(c) In case where molecular weight of the separate polypeptides are to be determined, both standard and unknown portions can be run on the same gel and the accuracy of results is much higher than disc-gel system.
Electrophoresis Apparatus:
The dimension of slab gels are 16 x 14 cm with 1.5 mm thickness containing 10 to 15 slots (25 x 8 x 1.5 mm) Glass plates are assembled and the separating gel recipes are poured between them, and left to polymerize for about 30-40 minutes.
Stack gel solution is then transferred to gel mould with a pasteur pipette. Combs are inserted and the stack gel is left to polymerize for about 20-30 minutes.
Sample Preparation:
Equal volume of 2x sample buffer is added to a protein sample (50 ml). 3 ml of 0.2% bromophenol blue dye is also added to it. The sample is heated in boiling water bath for 2 minutes and the protein denatures completely. This sample is then loaded to gel slot with micropipette. The electrophoresis run is done bromophenol blue dye reaches the bottom of the gel.
Staining is done by using dye as coomasic blue containing coomasic brilliant blue R 250 dissolved in 95% methanol and glacial acetic acid or silver staining containing 0.36% sodium hydroxide, ammonium hydroxide and 19.4% silver nitrate, citric acid, formaldehyde and 95% ethanol.
Data Recording:
After electrophoresis the glass plates are separated and the gel is kept on a clean glass plate. The length of the separation gel and the distance travelled by the bromophenol blue dye are measured. Staining and destaining is done. After destaining the gel is kept on top of a light box and the length of the separation gel and the distance travelled by each polypeptide are measured from top of separation gel.
The Rf of each polypeptide is calculated by:
Data Analysis:
After calculation of Rf values of each of the protein bands, a data matrix is usually prepared with two rows corresponding to the characters. The score could be ‘1’ for the presence and ‘O’ for absence of a band or polypeptide.
The data matrix thus prepared is ready for further analysis, essentially through numerical means to compute different similarity or dissimilarity indices and finally to construct a scheme of classification based on such protein characters.
Taxonomic Significance:
The taxonomic significance of the results of electrophoresis are useful mainly at and below generic levels. The most valuable results was obtained in cereals related to wheat in relation to the genomic constitution and ancestors of the tetraploids and Hexaploids.
Johnson worked on storage proteins and concluded that the hexaploid wheat (Triticum aestivum) contains a sum of protein possessed by the diploid species which are ancestors of it.
It had been studied in wild variety of Cajanus cajan while studying total seed storage protein in them SDS_PAGE method. Its protein profile of wild species were found to be very specific and distinct, pointing out towards the polyphyletic origin of the cultigen.
(c) Amino Acid Seauencins:
It is a modern way of identifying pure protein down to atomic level. First step is to break the total polypeptide chain into smaller peptide fragments and then they are sequenced separately.
Amino acid sequencing investigates the variation in the precise sequence of amino acids in a single homologous protein throughout a range of organisms. The process depends upon the fact that proteins do not have a single invariable structure, but a proportion of it may without altering its essential function.
Cytochrome ‘C’ is the molecule that was first used in plants for sequencing purpose. 79 out of 113 amino acids vary from species to species but alternation of even one of the other 34 destroys the functioning of the molecule. Stace (1989) determined amino acid.
Sequence of cytochrome in 25 species of vascular plants (fig.1). Sequence of Plstocyanin, Ferredoxin, Ribulose-I, 5-bis phosphate Carboxylase, etc. is also carried out in various plants. Plastocyanin sequence of over 70 species of angiosperms have been determined so far.
Amino acids sequences of protein are very useful taxonomically at the level of families and order as they are precise, relatively stable and independent of the traditional characters. The construction of a phylogenetic tree based on amino acid sequence and its using the ancestral sequences method permits the reconstruction of a precise, quantitative and objective topology of relationship.
